Currently, there are over 80,000 chemicals in use and approximately 2000 new chemicals are introduced into use every year. Regulatory agencies have recognized the need for alternative toxicological methods and models to decrease the time and expense of current toxicity testing protocols. In association with the National Toxicology Program (NTP), our group is developing C. elegans as an alternative organism for in vivo toxicological testing. Short life cycles, easy and inexpensive maintenance and culturing, and detailed biological knowledge has allowed for the development of rapid, low-cost toxicity tests that readily lend themselves to mechanistic studies of toxicant actions. Because of the evolutionarily conserved nature of the stress-response and other relevant pathways, it is likely that responses elicited in C. elegans will be applicable to understanding similar processes in higher organisms, including humans. This group is part of the National Toxicology Program and within the Biomolecular Screening Branch. The screening facility is involved in the development of C. elegans as an alternative organism for toxicological testing. There are two major activities of this group: research and development of C. elegans MTS and HTS technologies and participation in the MOU among the NTP, EPA, and NCGC, designated Tox21 (Science 15 February 2008 319: 906-907 DOI: 10.1126/science.1154619). Development of C. elegans medium-throughput screening assays - Protocols for the monitoring of growth, size, reproduction, feeding, and movement have been developed and have been used to test over 100 toxicants. Included in the creation of monitoring protocols, statistical analysis routines have been developed specifically for the C. elegans data. We have submitted one manuscript on the feeding assay and are completing additional studies that will be included in manuscripts currently being prepared on the reproduction and growth assays published manuscripts on the development and application of the feeding, reproduction, and growth assays as well as reviews on their combined use. In collaboration with Grace Kissling (EDBP), Marjolein Smith (SRA) we have developed a mathematical model that described C. elegans growth and the effects of toxicants on growth parameters. This year the WormTox group tested the chemicals in the ToxCAST phase 2 library (700), in addition to cadmium, mercurical, DMSO, paraquat, chlorpyrifos, MNNG and tunicamycin. We have also created an automated, high-content, high-throughput in vivo toxicological assay. Transcriptional response was measured in several strains of transgenic C. elegans containing fluorescent proteins driven by the promoters of stress-inducible C. elegans genes. This system captured both changes in pathway activation, as well as tissue-specificity of gene expression. Currently, we have 14 gene constructs available for testing -- ced-3, cyp-35A2, gcs-1, gst-38, gst-4, hsp-16.2, hsp-16.4, hsp-17, hsp-4, hsp-6, hsp-60, mtl-2, ugt-1 and ugt-13. Each transgenic strain has been confirmed to respond to at least one stimulus, usually heat shock; however, the reaction was often obscured by high baseline fluorescence levels. To select genes with a sensitive and specific stress response, we compared transcription of stress response genes using an array of well understood compounds -- paraquat, an oxidative stressor; N-methyl-N'-nitro-N-nitrosoguanidine, a DNA damaging agent; cadmium, a heavy metal toxin; chlorpyrifos, an organophosphate neurotoxin; tunicamycin, an endoplasmic reticulum stressor; and heat shock, a common C. elegans control stressor. By observing changes in fluorescence by microscope, we showed that at least one of the first 6 genes tested (cyp-35A2, gst-4, hsp-16.2, hsp-16.4, hsp-4, and hsp-60) upregulated in response to each toxicant. The response was generally mechanistically relevant; for example, hsp-4 (known to respond to endoplasmic reticulum stress) transgenic nematodes increased signal after tunicamycin treatment. The assay was optimized using pcyp-35A2::mCherry, punc-47::GFP nematodes treated with 6 concentrations of chlorpyrifos. This combination typified both changes in intensity and location of fluorescence. Florescence data were measured from images of transgenic nematodes taken with a high content imager and analyzed using CellProfilers WormToolbox. The software is a high-throughput imaging analysis program specifically designed for use with nematodes. Initial studies indicate that the results of the new assay are similar in trend, although less sensitive, than quantitative real-time polymerase chain reaction (qRT-PCR). Future studies will compare all transgenic nematodes currently available to the seven stressors by both qRT-PCR and the novel high-content, high-throughput assay. We are also developing a new assay to measure mitochondrial function by measuring ATP in vivo levels. Using a strain of C. elegans which constitutively expresses firefly luciferase, we are developing an assay that tracks levels of ATP pools as a marker for physiological status. Changes in luminescence following toxicant exposure will reflect changes in ATP levels. This assay will be used to determine the relative effects of a variety of chemicals on physiological health instead of the more common changes in morphological traits.